This proposal is aimed at developing Prussian blue nanoparticles (PBNPs) as a new generation of T1-weighted MRI contrast agents (CAs) with high relaxivity, long blood circulation times and ability to penetrate the cell membrane. Prussian blue (PB) is iron(III) hexacyanoferrate(II) with anidealized formula Fe4III[FeII(CN)6]3.nH2O (n=14-16) in which two different iron centers, Fe3+ (high-spin S=5/2) and Fe2+ (low-spin S=0) are bridged by the CN- groups. In the crystal structure of PB, a quarter (25%) of the FeII(CN)6 unit is absent from the crystal lattice, creating a large cavity inside the structure that is filled with water molecules. The missing FeII(CN)6 unit also causes the Fe3+ center to be coordinated by one water molecule and five CN- groups, thus giving rise to an active inner-sphere relaxation mechanism for enhancing the T1 relaxation. Due to the strong ligand-field effect and simultaneous coordination of the CN- group to both the Fe3+ and Fe2+ centers in the extended 3D network, the CN- ligand and the Fe3+/Fe2+ ions are completely locked in their lattice positions and cannot be released from the structure. As a result, PB has the lowest solubility product constant ever measured for any compound (Ksp=10-41). We have found that replacement of some of the Fe3+ ions with Mn2+ or/and Gd3+ ions in the crystal lattice can form the manganese- or gadolinium-incorporated nanoparticles, Mn@PBNPs and Gd@PBNPs with significantly increased r1 relaxivity. Besides, the structural rigidity and reduced tumbling rates of PBNPs in solution, as compared to the small molecular Gd3+-chelates, can contribute to additional T1-weighted MRI contrast enhancement in this new nanoplatform. Our goals are: (i) to explore methods for optimizing the r1 relaxivity by adjusting the nanoparticle size, level of Mn2+- or/and Gd3+-doping, and surface coating with small molecules or polymers;(ii) to systematically investigate the characteristics of cellular uptake and cellular imaging as well as potential for image-guided drug delivery applications;and (iii) to simultaneously incorporate Mn2+ or/and Gd3+ ions along with the radionuclide Ga-67 or Ga-68 for MRI-SPECT and MRI-PET bimodal imaging applications. We will endeavor to test the following four hypotheses: 1) Prussian blue nanoparticles, when properly tailored and engineered, will be effective in reducing the longitudinal relaxation time of protons from bulk water. Incorporation of Mn2+ or/and Gd3+ into this nanoplatform will significantly increase the r1 relaxivity;2) Prussian blue nanoparticles will be internalized by cells, exhibit no toxicity and be effective in cellular imaging and in delivering small-molecular agents;3) Prussian blue nanoparticles will be effective T1-weighted MRI contrast agents in vivo; and 4) Simultaneous incorporation of paramagnetic ions of manganese(II) or/and gadolinium(III) along with the radionuclide Ga-67 or Ga-68 into Prussian blue nanoparticles will produce effective bimodal contrast agents for in vitro and in vivo MRI-SPECT and MRI-PET imaging. Impact Our approach to exploring PBNPs as novel T1-weighted MRI is unprecedented and represents a paradigm shift in the design of new-generation CAs. The new paradigm that will emerge from this proposed research will prove to be revolutionary rather than evolutionary for increasing r1 relaxivity in a novel class of particulate T1-weighted MRI CAs, and thus will have high potential to produce a major breakthrough in MRI diagnostic imaging and may even completely change the landscape in this area of research. PUBLIC HEALTH RELEVANCE: Prussian Blue Nanoparticles as Cellular T1 MRI Contrast Agents We aim to develop novel Prussian blue nanoparticulate T1-weighted MRI contrast agents suitable for cellular imaging of cancer cells. Such agents will penetrate the cell membrane, exhibit no cytotoxicity, and can integrate imaging and delivering capabilities into a single platform for image- guided drug delivery. The design paradigm developed from this research will prove to be transformative and have potential to significantly impact the diagnostic MR imaging.